br Materials and methods BAECs
Materials and methods BAECs (CAB30405, Toyobo Life Science, Osaka, Japan) were cultured in Dulbecco’s Modified Eagle Medium supplemented with 10% Fetal Bovine Serum (04-001-1A-US, Biological Industries, Kibbutz Beit Haemek, Israel) and 1% antibiotic-antifungal agent containing 10,000-μg/mL penicillin, 10,000-μg/mL streptomycin, and 25-μg/mL amphotericin B. Cells were incubated at 37 °C with a humidity of 100% in an incubator containing 5% CO2 and 95% air (BNR-110, ESPEC, Osaka, Japan). All experiments were performed using BAECs that were cultured in plastic-based dishes (3910-035, IWAKI, Tokyo, Japan) and were harvested at passages of 7–10. We applied fluid shear stress and hypoxia loads using the following device. The flow circuit maintained airtightness in the flow circuit, and cultured cells were incorporated into the parallel plate-type flow chamber in dishes. The flow chamber comprised a parallel flat plate chamber, a flow chamber, a gasket (silicone sheet), and plastic HhAntag synthesis dish. The upper acrylic plate and the lower glass plate were separated by a 0.2-mm thick rectangular silicon sheet. Flow rates were adjusted using a peristaltic pump (EYELA: RP-1000, Tokyo Rikakikai Co, Tokyo, Japan) and pulses of flow from the pump were eliminated using a pulse damper. Media from the chamber were returned to a reservoir tank, and culture solutions were maintained in a constant temperature bath at 37 °C. The flow chamber was also placed in an insulated enclosure that was maintained at 37 °C using a temperature controller (TJA550P: As one, Tokyo, Japan). In this study, we investigated the phosphorylation of eNOS at Ser635 under the following 3 conditions; hypoxia, simultaneous shear stress and hyperoxia, and simultaneous shear stress and hypoxia. Shear stress was 2.0 Pa and the flow was static for non-shear stress experiment. Culture medium was incubated in a gas mixture of 5% CO2 and 95% air for hyperoxia experiments and 90% N2, 5% O2, and 5% CO2 (venous O2 level) for hypoxia experiments, respectively. To ensure that the cells were exposed by hypoxia for a short time, the medium was incubated thoroughly in the hypoxic gas before experiments. Flow inside the flow chamber was assumed steady, and shear stress on cells were controlled according to the following equation ,where μ is the viscosity coefficient of water at 37 °C (0.695 × 10−3 Pa s), h is the height of channel (0.27 × 10−3 m), and b is the channel width (14 × 10−3 m). After these exposures, cells were lysed using lysis buffer (Mammalian Cell Lysis Kit, Phosphatase Inhibitor Cocktail1, SIGMA, United States) and lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) after quantifying protein concentrations using a DC Protein Assay-Kit (BIO-RAD, California, United States). SDS-PAGE analyses were performed using Miniprothian3 cells (BIO-RAD, California, United States) and proteins were then transferred onto polyvinylidene difluoride (PVDF) membranes (BIO-RAD, California, United States) using a Criterion transblot cell (BIO-RAD, California, United States). Total and phosphorylated eNOS were then immunoblotted using primary anti-eNOS IgG (Santa Cruz Biotechnology, Texas, United States) and anti-phosphorylated eNOS IgG (Ser635) antibodies (Millipore, Darmstadt, Germany), followed by secondary biotin conjugated anti rabbit IgG (KPL, Milford, United States). PVDF membranes were also stained using an Amplified AP immunoblot Kit (BIO-RAD, California, United States) and anti-eNOS IgG and anti-phosphorylated eNOS IgG, which were applied at room temperature for 1 h at a dilution of 1:2000. After immunostaining, membrane images were generated using an LAS-4000 IR multicolor (FUJIFILM, Tokyo, Japan) and phosphorylated eNOS and eNOS band intensities were quantified using ImageJ . Digitized phosphorylated eNOS protein expression levels were normalized to those of total eNOS at each time point. Phosphorylated eNOS protein expression data from hypoxia, and a combination of shear stress and hyperoxia, and a combination of shear stress and hypoxia are reported as means ± standard errors of the mean (SE) from 5 individual experiments. Differences between treatments were identified using analysis of variance (ANOVA) with Tukey’s post hoc tests and were considered significant when p was <0.05.